The present MERIT Award extension will maintain its focus on the remarkable structural adaptability of apolipoproteins. This class of plasma protein performs essential functions, including receptor ligands, modulators of lipid metabolic enzymes and stabilization of plasma lipoproteins. Methods to introduce novel functional properties into reconstituted HDL (rHDL) will be exploited by altering rHDL lipids, introducing hydrophobic bioactive agents and tethering extraneous proteins. The therapeutic utility of these stable, self- assembled, water-soluble nanoscale size particles will be investigated. As a corollary to their physiological role in lipid transport and cholesterol efflux, the use of designer rHDL for replacement therapy applications and bioactive agent delivery is a rapidly advancing research front Our efforts are designed to validate concepts related to protein binding to rHDL and their delivery to cells. In aim 1, it is hypothesized that the Gaucher's disease phenotype in a macrophage cell model will be corrected by incubation with a phosphatidylserine-enriched, glucocerebrosidase-tethered rHDL. Enzyme uptake will be evaluated by flow cytometry / confocal fluorescence microscopy. Glucocerebrosidase activity in cell lysates, as well as changes in the cellular glucocerebroside content, will be measured. In Aim 2, we hypothesize that cellular delivery of Niemann-Pick C2 (NPC2) protein-tethered rHDL via apoE dependent uptake by members of the low-density lipoprotein receptor family will result in NPC2 delivery to lysosomes, rescuing the Niemann-Pick disease phenotype in GM18455 fibroblasts. NPC2-tethered apoE-containing rHDL will be incubated with cultured GM18455 fibroblasts and the ability of apoE to promote binding, uptake and lysosomal delivery of NPC2 will be determined, along with effects on lysosomal cholesterol content / organelle morphology. In Aim 3, agonist-induced activation of macrophage cholesterol efflux will be investigated. We hypothesize that one or more liver X receptor / retinoid X receptor (LXR / RXR) agonist can be delivered to macrophages in complex with rHDL. It is further hypothesized that nuclear hormone receptor activation will induce apoE / ABCA1 expression, resulting in increased cholesterol efflux capacity. The ability to generate water soluble, targeted rHDL capable of receptor-mediated protein / bioactive agent delivery to cells represents a novel strategy for disease treatment. The intrinsic versatility of the lipid and protein components of rHDL allows for their assembly with additional components, yielding stable complexes that can be used as delivery vehicles